This application is based on and claims priority of Japanese Patent Application No. Hei 11-196733 filed on Jul. 9, 1999, the content of which is incorporated herein by reference.
1. Field of the Invention
The present invention relates to a microscope. The present invention especially relates to a confocal microscope which properly measures a microscopic structure or a three dimensional structure of a sample.
2. Description of the Related Art
Traditionally, typical confocal microscopes have a disk that has a plurality of pinholes therein. For example, a disk called a ipkow-disk is used as the disk of a disk-scanning confocal microscope. The ipkow-disk has a plurality of pinholes, which are arranged in a spiral on the disk. Furthermore, pinholes of the ipkow disk are spaced a distance of about ten times the pinhole diameter. An example of a confocal microscope using an improved ipkow-disk is described by Juskaitis, T. Wilson et al. fficient real-time confocal microscopy with white light sources Nature Vol. 383, October 1996, pp. 804-806.
FIG. 10 shows a structure of the disk scanning confocal microscope described by T. Wilson et al. This disk-scanning confocal microscope uses a halogen lamp, a mercury lamp etc. as a light source 1. A collimating lens 2 and a PBS (polarizing beam splitter) 3 are disposed in the optical path of a light beam emitted from the light source 1. A sample 6 is disposed in a reflected light path of the PBS 3. The light beam is reflected by the PBS 3. Then the reflected light beam goes on to the sample 6 through a rotating disk 4 a quarter-wave plate 12 and an objective lens 5.
The collimating lens 2, the PBS 3, the quarter-wave plate 12 and the objective lens form an optical system to direct the light beam emitted from the light source 1 to the sample 6.
The rotating disk 4 is a random pinhole disk shown in FIG. 11. This disk has a random pinhole pattern portion 4a, an open portion 4b, and opaque portions 4c and 4d. The random pinhole pattern portion 4a has a plurality of pinholes, which are arranged on the disk. Furthermore, each pinhole of the disk is at a distance from another pinhole almost equal to the pinhole in diameter. The open portion 4b transmits the light beam emitted from the light source 1. The opaque portions 4c and 4d are disposed between the random pinhole pattern portion 4a and the open portion 4b. 
The rotating disk (rotating object) 4 is made of a transparent circular glass. Low-reflection films made of chrome films are deposited on the transparent circular glass to form opaque portions 4c and 4d. Likewise, the random pinhole pattern portion 4a is made by depositing the low-reflection film (chrome film etc.) on the transparent circular glass except at the location of pinholes. Therefore, each opaque portion 4c, 4d and the random pinhole pattern portion 4a except the pinhole locations will shade the light beam emitted from the light source 1. The rotating disk 4 is coupled to a rotating shaft 7, which is coupled to a shaft of a motor so as to rotate shaft 7 at constant speed.
The rotating disk 4 having the pinholes can be replaced with a line pattern disk shown in FIG. 12. The line pattern disk has a line pattern portion 4e instead of the random pinhole pattern portion 4a on the transparent circular glass. The line pattern portion 4e has a plurality of lines. These lines spaced an almost constant distance each other. These lines are made by depositing the low-reflection film (chrome film etc.) to shade the light beam emitted from the light source 1. That is, the line pattern portion 4e has alternate stripes of light-opaque portions and light-permeable portions (or light-semitransparent portion).
Returning to FIG. 10, a focusing lens 8 and a CCD camera 9 are disposed in a transmitted light path of the PBS 3. The reflected light beam returns toward the PBS 3, after the light beam emitted from the light source 1 is reflected by the sample 6. The reflected light beam passes through the PBS 3, and goes on to the CCD camera 9 through the focusing lens 8. An image output-terminal of the CCD camera 9 is connected to a computer 10 so as to capture an image. After capturing the image, the computer carried out an image processing so as to display the image on the monitor 11.
By using the above structure, the light beam emitted from the light source 1 is directed to the PBS 3 through the collimating lens 2. The light beam reflected by the PBS 3 becomes rays of light incident on the rotating disk 4 which is rotated at constant speed. The light beam passing through the random pinhole pattern portion 4a (or the line pattern portion 4e) or the open portion 4b of the rotating disk 4 becomes a circularly polarized light beam by passing through the quarter-wave plate 12. Then the circularly polarized light beam is focused on the sample 6 through the objective lens 5.
The light beam reflected by the sample 6 becomes a polarized light beam which is perpendicular to the light beam incident on the sample 6 by passing through the objective lens 5 and the quarter-wave plate 12. The polarized light beam passes through the random pinhole pattern portion 4a (or the line pattern portion 4e) or the open portion 4b of the rotating disk 4 again. Then the polarized light beam transits the PBS 3, and becomes rays of light incident on the CCD camera 9 through the focusing lens 8.
Now, a timing for taking an image by means of the CCD camera 9 must be synchronized with the rotating speed of the rotating disk 4 so as to capture a composite image and a bright field image. The composite image is captured when the light beam reflected by the sample 6 is passing through the random pinhole pattern portion 4a(or the line pattern portion 4e). The bright field image is captured when the light beam reflected by the sample 6 is passing through the open portion 4b. The composite image comprises a confocal image including non-confocal components. The bright field image comprises a non-confocal image.
That is, an image stored in the CCD 9 is captured in the computer 10 as a composite image during the time of the reflected light beam passing through just about a semi-circular area of the rotating disk 4 including the random pinhole pattern portion 4a(or the line pattern portion 4e). Next, an image stored in the CCD 9 is captured in the computer 10 as a bright field image during the time the reflected light beam is passing through just about another semi-circular area of the rotating disk 4 including the open portion 4b. 
After that, above two data captured sequentially are processed by a subtractive operation in the computer 10 so as to extract confocal components only. The confocal components are displayed on the monitor 11 as a confocal image. The following describes the subtractive operation with the computer 11.
(The composite image)xe2x88x92kxc3x97(the bright field image)=(The confocal image): k is constant
Therefore, where the CCD camera 10 is a progressive scanning camera, the composite image is captured while the rotating disk 4 makes a semi-circular rotation thereof. After that, the bright field image is captured while the rotating disk 4 makes a next semi-circular rotation thereof. That is, the composite image and the bright field image are captured sequentially. Then above two captured data are processed by a subtractive operation with the computer 10 so as to extract confocal image data. The confocal image is displayed on the monitor 11. The above mentioned sequence of processes is made sequentially, and enables to display the confocal image on the monitor 11.
On the other hand, in case where the CCD camera 10 is an interlaced system camera, an image, which includes a composite image is captured from even-numbered lines of the CCD camera 10 and a bright field image is captured from odd-numbered lines of the CCD camera 10. Therefore, the confocal image is extracted by subtracting odd-numbered lines from even-numbered lines.
As mentioned above, a conventional disk-scanning confocal microscope such as T. Wilson type has a structure to rotate the rotating disk 4 to capture the composite image by passing the light beam through the random pinhole pattern portion 4a or the line pattern portion 4e, to capture the bright field image by passing the light beam through transmitting portion (the open portion 4b), and to extract the confocal image by subtracting one of the composite image and the bright field image from the other image. That is, a confocal microscope such as T. Wilson type captures the composite image and the bright field image sequentially while the rotating disk 4 makes one rotation.
However, the above conventional confocal microscope extracts a confocal image from the composite image and the bright field image, which are captured sequentially in time when the rotating disk 4 makes one rotation. That is, one image (the confocal image) is determined from two images. In other words, since the display rate of the above conventional confocal microscope is a half its capturing rate, a real-time confocal image can not be displayed on the monitor smoothly. Furthermore, since the number of horizontal lines of the image are half of the number of CCD lines, a high resolution can not be obtained.
The present invention provides a confocal microscope which overcomes these problems. It has a light source emitting a light beam, an optical system for directing the light beam to the sample, a rotating object having a transparent portion transmitting the light beam emitted from the light source, and a light-permeable portion which has transparent and opaque areas. Also included is an element for capturing a composite image and a bright field image, the composite image including a confocal image and a non-confocal image. These are captured respectively, on the basis of the light beam passing through the light-permeable portion, and the bright field image captured on the basis of the light beam passing through the transparent portion. An operating element performs a subtraction between a newest captured image and a last image captured before the newest captured image so as to extract the confocal image whenever the operating element captures a newest image. A detecting part is disposed so as to detect whether a newest image being presently captured is the composite image or the bright field image. The composite image and the bright field image are captured alternately while the rotating object is rotating, and each of the newest captured image and the last image corresponds to either the composite image or the bright field image.